Particulate Protein Kinase C Research Articles

Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917–924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4–8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET(A) and ET(B)) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET(A) and ET(B) receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC alpha, beta2, delta, epsilon and zeta were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.

Protein kinase C distribution and translocation in rat myocardium: Methodological considerations

Introduction: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC distribution have not been systematically examined. Accordingly, we sought to determine whether homogenization methods (Polytron, glass–glass tissue grinder), detergent selection and concentration, or centrifugation protocols affect PKC (α, ɛ) distribution or phorbol-12-myristate-13-acetate (PMA)-induced translocation. Methods: Hearts of male F344 or Wistar rats were Langendorff perfused with either 100 nM PMA or vehicle, and LV cytosolic and particulate PKC (α, ɛ) distributions were assessed by differential centrifugation and Western blotting. Results: Following 100 000 × g centrifugation of the homogenate, resuspension of the pellet (P 1) in 0.1% sodium dodecyl sulfate (SDS) increased electrophoretic mobility of PKC (α, ɛ) such that PKCɛ comigrated with a nonspecific band. Resuspension of P 1 in Triton X-100 (TX) did not affect mobility but decreased P 1 PKC (α, ɛ) levels in a TX-concentration-dependent manner; however, this decrease was found to be due to differential protein solubilization. Decreased levels of PKC (α, ɛ) were also noted in soluble and P 2 (supernatant of 100 000 × g centrifugation of P 1) fractions due to increased Polytron burst and total homogenization times. Interestingly, the P 2 fraction also revealed Polytron-dependent decreases (47% vs. glass–glass tissue grinder; p<0.05) in PKCɛ following an initial 1000 × g centrifugation and an increased PMA-dependent translocation of PKC (α, ɛ; 2.4-fold and 1.6-fold, respectively, vs. P 1; p<0.05). Discussion: Taken together, these results suggest that PKC isolation procedures should be carefully considered when designing or comparing LV PKC studies due to the potential effects isolation may have on PKC distribution and translocation.

Effects of fenfluramine and antidepressants on protein kinase C activity in rat cortical synaptoneurosomes.

Fenfluramine releases serotonin (5-HT) via the 5-HT transporter (SERT). Previous work has shown that amphetamine increases particulate protein kinase C (PKC) activity in striatal synaptoneurosomes. The increased PKC activity is linked to the outward transport of dopamine, and when release is diminished, the inward transport of amphetamine inhibits PKC instead. Since there is homology among monoamine transporters, this study was undertaken to determine if D-fenfluramine has similar effects on PKC. The role of 5-HT receptors and endogenous 5-HT were also examined. Naive rats and rats pretreated with p-chlorophenylalanine (PCPA), a 5-HT synthesis inhibitor, were sacrificed. Cortical synaptoneurosomes were prepared and incubated with fenfluramine. PKC activity was determined by thiophosphorylation of endogenous substrates. It was found that 5-HT, D/L-fenfluramine, and D-fenfluramine increased PKC activity in a time- and dose-dependent manner. The 5-HT-mediated increase in PKC activity was attenuated by pretreatment with the 5-HT(2) antagonist ketanserin, but not with the SERT inhibitor fluoxetine. The D-fenfluramine-induced increase in PKC activity was completely prevented, however, by pretreatment with SERT inhibitors and partially with ketanserin. It was also attenuated by pretreatment with PCPA, resulting in a dose-dependent inhibition of PKC instead. Thus, when 5-HT release was diminished the uptake of D-fenfluramine inhibited PKC. Similar effects have been observed with amphetamine. Unlike D-fenfluramine, the D/L-fenfluramine-induced increase in PKC activity was partially resistant to PCPA pretreatment but was attenuated with bupropion, a dopamine transporter (DAT) inhibitor. SERT inhibitors (sertraline, paroxetine, citalopram, and fluoxetine) also increased PKC activity. Nefazodone and bupropion increased PKC activity, but mirtazapine was relatively inactive. The SERT inhibitor-induced increase in PKC was unaffected by pretreatment with PCPA but was inhibited by calcium. Similar effects on PKC activity have been observed with DAT inhibitors. These results, showing that D-fenfluramine altered PKC activity similar to D-amphetamine, suggest that the topographic homology between DAT and SERT may extend to their effects on PKC activity.

Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells.

We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin-Darby canine kidney cells. We utilized beta-chloroalanine (BCA) to inactivate serine C -palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [(32)P]P(i), [(3)H]palmitic acid and [ methyl -(3)H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [(3)H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous D-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells.

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

Carvedilol inhibits pressure-induced increase in oxidative stress in coronary smooth muscle cells.

The cellular mechanisms by which hypertension enhances atherosclerosis are still not known in detail. Recently, evidence has been obtained that oxidative stress plays a role in the pathogenesis of pressure-induced atherosclerosis. We examined the effects of pressure on oxidative stress in cultured human coronary smooth muscle cells (SMCs). Application of increased pressure (+100 mmHg) with He gas for 48 h increased oxidative stress of measured by flow cytometry by 71% and F2-isopretane by 77%. Increased pressure also increased the activities of phospholipase D (PLD), and particulate protein kinase C (PKC). The PLD inhibitor suramin 100 micromol/l, 1-butanol 40 mmol/l, and the PKC inhibitors chelerythrine 1 micromol/l and calphostin C 100 nmol/l and completely blocked the increase in oxidative stress induced by pressure. Carvedilol 1 micromol/l but not propranolol 1 micromol/l blocked pressure-induced increases in oxidative stress in cultured SMCs. These findings suggest that pressure increases oxidative stress and that carvedilol significantly inhibits pressure-induced increase in oxidative stress in cultured human coronary smooth muscle cells.

Modulation of radiation-induced protein kinase C activity byphenolics

Natural phenolic compounds were tested in vitro for their effect onthe activity of protein kinase C (PKC) isolated from the liver cytosol and theparticulate fraction of unirradiated mice and mice irradiated at 5 Gy.Following irradiation, the PKC activity was found to be increased in bothcytosolic and particulate fractions. Curcumin, ellagic acid and quercetin wereeffective in inhibiting radiation-induced PKC activity. Curcumin and ellagicacid were found to be more inhibitory towards radiation-induced PKC activity,while quercetin was the least effective. Curcumin was found to inhibit theactivated cytosolic and particulate PKC at very low concentrations. Activationof PKC is one of the means of conferring radioresistance on a tumour cell.Suppression of PKC activity by phenolics may be one of the means of preventingthe development of radioresistance following radiotherapy.

Inhibition by Eicosapentaenoic Acid of Oxidized-LDL- and Lysophosphatidylcholine-Induced Human Coronary Artery Smooth Muscle Cell Production of Endothelin

The objectives of the present study were (1) to determine whether oxidized low-density lipoprotein (LDL) and lysophosphatidylcholine (lyso-PC), a major phospholipid component of oxidized LDL, stimulate the production of endothelin-1 (ET)-1 in cultured human coronary artery smooth muscle cells (SMCs), and (2) to examine the possible effect of an antiatherogenic agent, eicosapentaenoic acid (EPA), on oxidized-LDL- and lyso-PC-stimulated ET-1 production in these cells. Oxidized LDL (10–50 µg/ml) and lyso-PC (10^–7 to 10^–5 mol/l) stimulated ET-1 production in a concentration-dependent manner. By contrast, the effects of native LDL and phosphatidylcholine were modest or absent. Lyso-PC (10^–7 to 10^–5 mol/l) and oxidized LDL (10–50 µg/ml) significantly induced particulate protein kinase C (PKC) activation. Lyso-PC- and oxidized-LDL-stimulated ET-1 production was significantly inhibited by PKC inhibitor, PKC (19–36). EPA (80–160 µmol/l) clearly suppressed ET-1 production stimulated by oxidized LDL and lyso-PC in a concentration-dependent manner. Furthermore, EPA (160 µmol/l) significantly inhibited lyso-PC (10^–5 mol/l)- and oxidized LDL (50 µg/ml)-induced particulate PKC activation. Results suggest that oxidized LDL and lyso-PC stimulate ET-1 production by a mechanism involving activation of PKC, and that EPA suppresses ET-1 production stimulated by lyso-PC as well as oxidized LDL probably through the modulation of PKC in human coronary artery SMCs. EPA may exert an antiatherosclerotic effect, in part, through these mechanisms.

κ -Opioid Receptor Stimulation Induces Arrhythmia in the Isolated Rat Heart via the Protein Kinase C/Na+–H+Exchange Pathway

The present study attempted to determine whether the protein kinase C (PKC)/Na+–H+exchange (NHE) pathway would mediate the arrhythmogenic action ofκ -opioid receptor (OR) stimulation. We first determined the effects of U50,488H, a selective κ -OR agonist, on PKC activity and cardiac rhythm in the isolated perfused rat heart, and intracellular pH (pHi), and Ca2+([Ca2+]i) and Na+([Na+]i) concentrations in the isolated ventricular myocyte. At 5–40μ m U50,488H concentration dependently increased the particulate PKC activity and pHi, and induced arrhythmia. 40 μ m U50,488H also increased [Na+]iand [Ca2+]i. The arrhythmogenic effects of 40 μ m U50,488H were abolished by nor-binaltorphimine, a selective κ -OR antagonist. Blockade of PKC and NHE with respective blockers, 1 μ m bisindolylmaleimide I or 0.5 μ m calphostin C, and 1 μ m 5-[N -methyl- N -isobutyl]amiloride or 1 μ m 5-([N -ethyl- N -isopropopyl]amiloride, abolished and significantly attenuated, respectively, the effects of κ -OR stimulation on pHi, [Na+]iand [Ca2+]i, and arrhythmia. To determine the role of pHi, we observed U50,488H-induced arrhythmia at pHi6.8. At this pHi, the pHiincreased gradually both in the presence and absence of 40 μ m U50,488H to a similar extent. While the increase in response to U50,488H was significantly less at pHi6.8 (from 0.09 to 0.10) than that at pHi7.1 (from 0.01 to 0.18), the arrhythmia induced by the agonist was the same at both high and low pHs. On the other hand, 5μ m monensin, a sodium ionophore, increased [Na+]iand [Ca2+]i, and induced arrhythmia to similar extents as U50,488H. PKC and NHE inhibitors, that significantly attenuated the effects induced by U50,488H, had no effect on those induced by monensin. In conclusion, κ -OR stimulation induces arrhythmia via PKC/NHE. [Na+]iand [Ca2+]i, but not pHi, may be directly responsible for arrhythmia induced byκ -OR stimulation.

Modulation of NDEA activated ras expression and protein kinase C activity by nicotinamide

Nicotinamide, an amide derivative of vitamin B 3, was found to inhibit the increase in ras expression, protein kinase C (PKC) activity, and diacylglycerol (DAG) levels in rats treated with N-nitrosodiethylamine (NDEA). It effectively modulated the carcinogen induced increase in the cytosol and the particulate PKC activities but not the nuclear PKC activity. The DAG levels were also modulated by the endobiotic. It may serve as an ideal modulator of carcinogenesis. Nicotinamide is currently under trial as a radio sensitizer, and inhibition of these parameters may be one of the ways in which it confers radiosensitivity.

Effect of age on calcium-dependent proteins in hippocampus of senescence-accelerated mice

The senescence-accelerated P8 mouse (SAMP8) is a well-characterized model for the age-related decline in acquisition and retention. Calcium-dependent protein kinase C (PKC) and calcium–calmodulin-dependent protein kinase (CAM K) have been implicated in these processes in the hippocampus. Therefore, the expression of hippocampal PKC and CAM K was determined in SAMP8 mice aged 4, 8, and 12 months. As measured by Western blotting, total hippocampal PKC-gamma protein declined linearly with age. In addition, the distribution of the PKC-gamma also changed with age. The amount of PKC in the particulate fraction declined linearly with age relative to the soluble PKC. The decline in total PKC and particulate PKC correlated with the previously reported decline in retention but not with the decline in acquisition. Western blotting revealed no consistent change in CAM KII protein levels. In addition to protein levels, Ca-dependent protein kinase activity may also be affected by changes in intracellular Ca concentration. Therefore, the levels of calbindin and the plasma membrane Ca pump, two proteins involved in maintaining low levels of intracellular Ca, were measured in the hippocampus. Calbindin protein declined progressively with age, but there was no significant change in total plasma membrane Ca pump expression. These studies demonstrate a decrease in the amount and distribution of hippocampal PKC-gamma in the SAMP8 between 4 and 12 months that is associated with decreased retention.

Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation: II. Activation and involvement of the alpha, epsilon, and zeta isoforms of PKC.

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Involvement of protein kinase C in the nitric oxide-mediated peripheral nerve disturbance in endotoxemic rats

The role of protein kinase C (PKC) in nitric oxide (NO)-mediated peripheral nerve disturbance in lipopolysaccharide (endotoxin, LPS)-treated rat was studied. The impaired Na+,K+-ATPase activities in sciatic nerves from LPS-treated rats were prevented by aminoguanidine (NO synthase inhibitor) and corrected by PKC agonist in vitro. Using Western blot to determine PKC isoforms α and β polypeptide levels in LPS-treated rat sciatic nerves, we found that α isoform was markedly reduced in the particulate fraction, but the β isoform was unaffected. The α and β isoforms in the cytosolic fractions were not significantly different as compared with control. This diminished particulate PKC α isoform was prevented by the treatment of aminoguanidine. Moreover, the motor nerve conduction velocity was significantly reduced in endotoxemic rats and corrected by aminoguanidine. These results indicate that the alteration of PKC α isoform in Na+,K+-ATPase-enriched fraction of sciatic nerve may be related to the NO-mediated peripheral nerve disturbance in endotoxemic rats.

Regulation of renal Na-HCO3 cotransporter: VIII. Mechanism Of stimulatory effect of respiratory acidosis.

We examined the effect of respiratory acidosis on the Na-HCO3 cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO2 for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO2 showed a significant increase in Na-HCO3 cotransporter activity (expressed as % of control levels, 5 min: 142 +/- 6, 2 hr: 144 +/- 13, 4 hr: 145 +/- 11, 24 hr: 150 +/- 15, 48 hr: 162 +/- 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO2 for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO2 for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO2 for 5 min or 4 hr. In cells treated with vehicle, 10% CO2 significantly increased cotransporter activity as compared to control cells exposed to 5% CO2. This stimulation by 10% CO2 was completely prevented by HAC or DHC at 5 min (5% CO2: 1.8 +/- 0.2, 10% CO2: 2.6 +/- 0.2, 10% CO2 + HAC: 1.6 +/- 0.2, 10% CO2: +DHC: 2.0 +/- 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO2 for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO3 cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis.

Effects of the peroxisome proliferator ciprofibrate and prostaglandin F2 alpha combination treatment on second messengers in cultured rat hepatocytes.

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatic tumors in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including eicosanoids. Peroxisome proliferators transiently induce increased cell proliferation in vivo. However, peroxisome proliferators are weakly mitogenic and are not co-mitogenic with epidermal growth factor (EGF) in cultured hepatocytes. Earlier study found that the peroxisome proliferator ciprofibrate is comitogenic with eicosanoids. In order to study possible mechanisms of the comitogenicity of peroxisome proliferator ciprofibrate and eicosanoids, we hypothesized that the co-mitogenicity may result from synergistic or additive increases of second messengers in mitogenic signal pathways. We therefore examined the effect of the peroxisome proliferator ciprofibrate, prostaglandin F2 alpha (PGF2 alpha) and the combination of ciprofibrate and PGF2 alpha with or without growth factors on the protein kinase C (PKC) activity, and inositol-1, 4, 5-triphosphate (IP3) and intracellular calcium ([Ca2+]i) concentrations in cultured rat hepatocytes. The combination of ciprofibrate and PGF2 alpha significantly increased particulate PKC activity. The combination of ciprofibrate and PGF2 alpha also significantly increased EGF, transforming growth factor-alpha (TGF-alpha) and hepatic growth factor (HGF)-induced particulate PKC activity. The combination of ciprofibrate and PGF2 alpha greatly increased [Ca2+]i. However, the increases of PKC activity and [Ca2+]i by ciprofibrate and PGF2 alpha alone were much smaller. Neither ciprofibrate or PGF2 alpha alone nor the combination of ciprofibrate and PGF2 alpha significantly increased the formation of IP3. The combination of ciprofibrate and PGF2 alpha, however, blocked the inhibitory effect of TGF-beta on particulate PKC activity and formation of IP3 induced by EGF. These results show that co-mitogenicity of the peroxisome proliferator ciprofibrate and eicosanoids may result from the increase in particulate PKC activity and intracellular calcium concentration but not from the formation of IP3.

Role of Ca(2+)-independent PKC in alpha 1-adrenoceptor-mediated inotropic responses of neonatal rat hearts.

We investigated the role of protein kinase C (PKC) in alpha 1-adrenoceptor (alpha 1-AR)-mediated positive inotropic responses of neonatal rat myocardium. Bisindolylmaleimide (BIM), an inhibitor of all PKC isoforms, reduced the positive inotropic responses to phenylephrine and methoxamine but not to isoproterenol. The positive inotropic effect of phenylephrine was not attenuated by Gö-6976, a selective inhibitor of Ca(2+)-dependent isoforms; it was potentiated by the 1,2-diacylglycerol kinase inhibitor R-59949. Phorbol 12,13-dibutyrate, an activator of both Ca(2+)-dependent and-independent PKC isoforms, as well as thymeleatoxin, a selective activator of Ca(2+)-dependent PKC isoforms, inhibited myocardial contractions, which were prevented by BIM and Gö-6976. BIM inhibited the phenylephrine-induced increase in particulate PKC activity but not the increase in phosphatidylinositide turnover. Phenylephrine induced translocation of only Ca(2+)-independent PKC-epsilon and -delta. These results suggest that activation of Ca(2+)-independent PKC isoforms by alpha 1-AR agonists plays a key role in alpha 1-AR-mediated positive inotropic effect on neonatal myocardium.

Alterations in the expression and cellular localization of protein kinase C isozymes epsilon and theta are associated with insulin resistance in skeletal muscle of the high-fat-fed rat.

We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with the development of insulin resistance in skeletal muscles from the high-fat-fed rat. Lipid measurements showed that triglyceride and diacylglycerol, an activator of PKC, were elevated four- and twofold, respectively. PKC activity assays indicated that the proportion of membrane-associated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC alpha, epsilon, and zeta levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC epsilon in red muscles from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC theta was downregulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamped rats increased the proportion of PKC theta in the particulate fraction of red muscle, this was potentiated by fat-feeding, suggesting that the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC epsilon and theta measurements from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, which was not elevated in fat-fed rats, suggesting that they were not simply a consequence of hyperglycemia. Our results suggest that these specific alterations in PKC epsilon and PKC theta might contribute to the link between increased lipid availability and muscle insulin resistance previously described using high-fat-fed rats.

Activation of protein kinase C during newt limb regeneration: effect of denervation.

Blastema cell proliferation during newt limb regeneration is a nerve-dependent process. The present study was undertaken to determine whether or not that process is mediated by protein kinase C (PKC) activation during limb regeneration in Pleurodeles walt. Analysis included evaluation of PKC activity and its subcellular localization at various stages of regeneration, both in vivo and in vitro. The data reveal an increase in PKC activity in both the cytosol and particulate fractions of whole blastemas reaching a maximum at the mid-bud stage, which correlates with blastema cell proliferation rate. Denervation significantly reduces blastema cell proliferation and also causes a reduction in membrane-associated PKC activity. The effect of PKC activity appears to be restricted to the blastemal mesenchyme, which exhibits a dramatic reduction in activity 96 h after denervation. In contrast, PKC activity in the epidermal cap did not change. Cultured whole blastemas likewise express a decrease in particulate PKC activity and therefore mimic denervated blastemas in this parameter. Co-culture of blastemas with spinal ganglia partially reduces the decline in PKC activity, and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a direct activator of PKC, also prevents the fall in membrane-bound PKC activity while stimulating blastema cell proliferation, in vitro. These data indicate that blastema cell (mesenchyme) proliferation is related to increased PKC activity and that PKC may therefore be involved in the nerve-dependent signalling pathway regulating the early phase of urodele limb regeneration.